The physiological cellular response to tissue injury in the skin progresses through a sequence of structured phases and normally results in a nearly complete recovery the injured area. Wounds can be either acute or chronic with respect to healing. In chronic wounds, the duration of the wound healing processes is either much slower or static. Wound healing depends on several factors, including the patient's age and physical condition, the location of the wound, the cause of the injury, and accompanying diseases such as diabetes or renal insufficiency, which all have a negative effect on wound healing processes.
Wound healing involve several populations of cells (thrombocytes or platelets, neutrophile granulocytes, macrophages, fibroblasts, and keratinocytes), soluble factors (cytokines and growth factors), and proteases (e.g., matrix metalloproteinases [MMPs], plasmin, and elastase). Healing initially involves hemostasis initiated by the activation of the clotting cascade. Fibrin clots forming the provisional wound matrix entrap erythrocytes and platelets and block blood flow. Numerous growth factors (e.g., platelet-derived growth factor (PDGF), platelet-derived angiogenic factor (PDAF), transforming growth factor and epidermal growth factor (EGF)) are released from platelet granules and chemotactically attract neutrophils, fibroblasts, endothelial cells, and keratinocytes into the wound. The initial release of growth factors from platelets is important in initiating the phases of wound healing.
Inflammation is the initial response to tissue injury. Within 6 h after tissue injury, inflammation begins. The main goal of the inflammatory phase is to provide rapid hemostasis and begin the sequence of events that leads to regeneration of tissue. Neutrophil granulocytes typically appear in wounds first and control bacterial contamination and cleanse the wound from cell detritus. After 48 h, the concentration of neutrophil granulocytes is maximized. Monocytes begin infiltrating the wound site 24 h after injury, attracted by chemotactic factors including complement factor 5, fibrin degradation products, and TGF-β. In response to wound cytokines, monocytes differentiate into wound macrophages to aid wound repair.
During the proliferative phase, the damaged, necrotic tissue that is being removed via phagocytosis starts to be replaced with living tissue that is specific to the local tissue environment. Proliferation is primarily characterized by granulation tissue. MMPs take part in the structured development of granulation tissue by removing damaged matrix proteins, helping cells migrate into the wound, and developing new blood vessels.
About 2 days after injury, macrophages from monocytes begin expressing growth factors. Macrophages continue to release PDGF, macrophage angiogenesis factor, and TGF-β. PDGF, macrophage angiogenesis factor, and angiotensin stimulate new blood vessel formation, generating granulation tissue in the wound. EGF, keratinocyte growth factor, and PDGF stimulate epidermal cells to migrate, divide, and differentiate (keratinize), covering the granulation tissue with a cellular barrier to desiccation and infection.
During remodeling, newly generated tissue reshapes and reorganizes to more closely resemble the original tissue. Remodeling begins about the 7th day of wound healing and can continue for 6 months to a year. Early in the remodeling, the provisional wound matrix, predominately fibrin and fibronectin, is replaced with proteoglycan molecules and collagen molecules (type III, type I) that become cross-linked by enzymatic action, which greatly increases the tensile strength of the scar matrix. In addition, some fibroblasts are stimulated to transform into myofibroblasts that contract the wound matrix. The high density of new blood vessels and myofibroblasts in the scar then decrease as vascular endothelial cells and fibroblasts undergo apoptosis, and the hypertrophic epidermal layer becomes thinner. At the end of the wound healing process, the wound is closed. However, the repaired tissue does not completely regenerate the original tissue structure, and some level of functionality of the scar tissue is usually lost.
Platelets play a prominent role as one of the first responders during the acute inflammatory phase. In response to tissue damage, platelets are activated resulting in the formation of a platelet plug and blood clot for hemostasis. The alpha granules of activated platelets contain numerous proteins that influence wound healing. Thrombin production ultimately occurs and converts fibrinogen to fibrin which binds to platelet surface receptors. Proteins from platelet degranulation are partly responsible for cellular chemotaxis, proliferation, and differentiation. This includes removal of tissue debris, angiogenesis, establishing the extracellular matrix, and regeneration of the appropriate type of tissue.
Platelet rich plasma (PRP) contains clotting factors and higher concentration of platelets than baseline. The portion of plasma that remains deficient in platelets is known as platelet poor plasma (PPP). PPP has clinical roles as fibrin sealant for hemostasis.
A common characteristic of chronic wounds is elevated protease activities. Thus, local (or systemic) treatment of chronic wounds with protease inhibitor(s) could promote healing. However, high levels of protease activity in chronic wounds of widely differing aetiology have been shown and may be related to a problem with the healing process itself rather than with the aetiology of the wound.
MMPs play vital roles in initial wound debridement as well as in the phases of angiogenesis, epithelialization, and scar remodeling. A balance between proteases and their inhibitors is necessary for a correct wound healing, and elevated levels of proteases and reduced levels of inhibitors have been found in chronic wounds. Increased levels of MMP-2 and MMP-9 have been demonstrated in various chronic wound liquids. Increased levels of MMP-1 and MMP-8 have been found in decubital ulcers, and MMP-13 in venous ulcer lesions. Reduced levels of TIMPs have been found in chronic wound fluid. The MMP-9 to TIMP-1 ratio may be a predictor for chronic wound healing, as an inverse correlation with the healing tendency of chronic pressure ulcers has been shown. (Ladewig et al).
Similar processes may occur in non-healing or poorly healing diabetic foot lesions. Loots et al., Dahn et al., Mansbridge et al.). Higher concentrations of MMPs and reduced concentrations of MMP inhibitors have been found in diabetic wounds compared with trauma lesions of a control group. Unlike normal wound healing, an overexpression of these proteases seems to support a delayed wound healing and lead to a failure of wounds to heal. Additionally, an imbalance between MMPs and TIMPs that contributes to the pathogenesis of nonhealing chronic lesions may exist. Chronic diabetic foot ulcers have been treated with the antibiotic doxycycline, which is also a competitive inhibitor of certain metalloproteases. Dressings that contain high concentrations of gelatin, which is a substrate for MMPs, have also been used. Elastase and plasmin activities in wound fluids have been found at significantly reduced by a local therapy with Promogran, which may improve healing by reducing the activities of MMPs in the molecular environment of the wound. Cullen et al. A dressing consisting of metal ions and citric acid has also been used and reduced reactive oxygen species and MMP-2 production in vitro.
Use of recombinant PDGF (Regranex) for diabetic foot syndrome showed improvements in the probability of healing and reduction of healing time. Smiell et al. It was also determined that the wound bed needed to be properly debrided for the growth factor to have maximum benefit. Thus, wound bed preparation is important, and emphasizes the removal of barriers to healing and the integration of advanced technologies in wound care.
Alpha-2-macroglobulin (A2M) is a highly conserved protease inhibitor present in plasma at relatively high concentrations (0.1-6 mg/ml). It is unique in its ability to inhibit all the major classes of proteases (Bhattacharjee et al., J. Biol. Chem. 275, 26806-11 (2000)). A2M can be produced by several cell types, such as hepatocytes, lung fibroblasts, macrophages, astrocytes and tumor cells (Borth W, Ann. N.Y. Acad. Sci. 737:267-72 (1994)). A2M often exists as a tetramer of four identical 180 kDa subunits that forms a hollow cylinder-like structure. It can present multiple target peptide bonds to attacking proteases in its central “bait” domain. A2M can be the major protease inhibitor acting on foreign proteases, such as snake venoms. However, there are many other protease inhibitors in the circulation and it has been proposed that A2M can have other functions including binding to and regulation of cytokine and growth factor activity, promotion of tumoricidal capabilities of macrophages, and enhancement of antigen presentation. A2M can also be a targeting carrier for cytokines or growth factors.
Despite advances in the understanding of the principles underlying the wound healing process, there remains a significant unmet need for suitable therapeutic options for wound care and tissue repair and improving and/or promoting wound healing, including wounds that do not heal at expected rates, such as delayed-healing wounds, incompletely healing wounds, and compromised wound healing such as is seen in chronic wounds, scarring and abnormal or excessive scarring, including keloid and hypertrophic scarring, atropic scarring, widespread scarring, and scar contractures, as well as adhesions including surgical adhesions. There is a need in the art for improved methods and compositions for treating conditions such as those caused by acute and chronic wounds, inflammation, fibrosis, scarring, and adhesions.
Therefore, it is an object of the invention to provide compositions, systems, methods, and kits for the detection, diagnosis, and treatment of inflammation, degradation of extracellular matrix, and wounds. It is another object of the invention to provide systems and methods to produce compositions for the treatment of inflammation, degradation of extracellular matrix, and chronic wounds. It is another object of the invention to provide biomarkers and methods for identifying sites of chronic wounds. It is another object of the invention to provide methods for diagnosing or assisting in the diagnosis of the presence of pathologies that are causative of chronic wounds. Yet another object of the invention is to provide biomarkers and methods to determine an appropriate therapy for a subject experiencing chronic wounds. Another object of the invention is to provide biomarkers and methods to monitor and assess the efficacy of a treatment for chronic wounds. Another object of the invention is to provide compositions and methods for treating chronic wounds and for selecting treatment sites and methods for treatment of chronic wounds.
It is another object of the invention to provide variant polypeptides for treating chronic wounds. It is another object of the invention to provide variant A2M polypeptides with a higher protease inhibitory activity than a wild-type A2M polypeptide. It is another object of the invention to provide methods of making variant polypeptides for the treatment of chronic wounds.